The present invention relates to fluorescein derivatives which are useful as agents for measurement of nitrogen monoxide. It also relates to agents for measuring nitrogen monoxide which comprise said compound.
Nitrogen monoxide (NO) is an unstable radical having a short life, and it has been elucidated that nitrogen monoxide has important functions as a physiologically active substance in a living body (featured in Gendai Kagaku (Chemistry Today), April, 1994). Methods for measuring nitrogen monoxide are mainly classified into indirect methods where oxidative degradation products of nitrogen monoxide such as NO2xe2x88x92 or NO3xe2x88x92 are measured, and methods where nitrogen monoxide is directly measured. The direct methods have been focused from the standpoint that they achieve detection and quantification of nitrogen monoxide under physiological condition. However, no measuring method has been developed to date that has sufficient specificity and high sensitivity and is applicable to an in vitro system.
For example, a chemiluminescence method which utilizes luminescence emitted during an ozonic oxidation of NO radicals (Palmer, R. M., et al., Nature, 327, pp.524-526, 1987); a method which comprises the step of measuring an absorption spectrum of metHb that is produced by an oxidation of oxyhemoglobin (O2Hb) (Kelm, M., et al., Circ. Res.66, pp.1561-1575, 1990); a method which comprises the step of measuring electric current generated during an oxidation by means of electrodes that are inserted into a tissue (Shibuki, K., Neurosci. Res.9, pp.69-76, 1990; Malinski, T., Nature, 356, pp.676-678, 1992); and the Griess reaction method (Green, L. C., et al., Anal. Biochem., 126, pp.131-138, 1992) are known as typical methods (as reviews, see, xe2x80x9c3. Method for measurement of NO,xe2x80x9d by Tetsuo Nagano, pp.42-52, xe2x80x9cApproach from the Latest Medicine 12, NOxe2x80x9d edited by Noboru Toda, published by Medical View Co., Ltd.; and Archer, S., FASEB J., 7, pp.349-360, 1993).
The Griess reaction method comprises a detection step that utilizes azo coupling between naphthylethylenediamine and a diazonium salt compound formed with NO2xe2x88x92 which is generated by the oxidation of nitrogen monoxide radicals. This method is advantageous because it does not require particular apparatuses or techniques, although nitrogen monoxide radicals are not directly measured by the method. In addition, NO3xe2x88x92 can also be measured after being reduced to NO2xe2x88x92 by using cadmium (Stainton, M. P., Anal. Chem., 46, p.1616, 1974; Green, L. C., et al., Anal. Biochem., 126, pp.131-138, 1982) or hydrazine (Sawicki, C. R. and Scaringelli, F. P., Microchem. J., 16, pp.657-672, 1971), and accordingly, the method also has characteristic feature that it enables the measurement of metabolites related to nitrogen monoxide.
2,3-Diaminonaphthalene has also been known as an agent for measuring nitrogen monoxide by detecting NO2xe2x88x92, as in a similar manner to Griess reaction method. This agent reacts with NO2xe2x88x92 under an acidic condition to form a fluorescent adduct, i.e., naphthalenetriazole (chemical name: 1-[H]-naphtho[2,3-d]triazole) (Wiersma, J. H., Anal. Lett., 3, pp.123-132, 1970). Details of the reaction conditions of 2,3-diaminonaphthalene and NO2xe2x88x92 have been studied, and it has been found that the reaction proceeds most rapidly at a pH not higher than 2, and completes in about 5 minutes at room temperature (Wiersma, J. H., Anal. Lett., 3, pp.123-132, 1970; Sawicki, C. R., Anal. Lett., 4, pp.761-775, 1971). The resulting adduct emits fluorescence most efficiently at a pH not lower than 10 (Damiani, P. and Burini, G., Talanta, 8, pp.649-652, 1986).
The method for measuring nitrogen monoxide using the above 2,3-diaminonaphthalene has characteristic features of 50- to 100-fold higher sensitivity compared to the Griess reaction method, since its detection limit is as low as approximately several tens nM (Misko, T. P., Anal. Biochem. 214, pp.11-16, 1993). This method is highly advantageous because it needs no particular apparatus or technique and can be carried out conveniently (as a review of the aforementioned method, see, DOJIN News. No. 74, Information, xe2x80x9cAn agent for the determination of NO: 2,3-diaminonaphthalene,xe2x80x9d Dojindo Laboratories Inc., 1995). However, the method does not utilize nitrogen monoxide, per se, but utilizes an oxidation product, i.e., NO2xe2x88x92, as a reactant. Accordingly, the method is considered as an indirect method when compared to those including direct measurement of nitrogen monoxide. Furthermore, because the reaction of 2,3-diaminonaphthalene with NO2xe2x88x92 is carried out under a strongly acidic condition (pH not higher than 2), the method has a problem in that it cannot be employed for detection or quantification of nitrogen monoxide under a physiological condition.
The inventors of the present invention conducted researches to provide a means that enables direct and highly sensitive measurement of nitrogen monoxide under a physiological condition, and as a result, they found that nitrogen monoxide can efficiently react with 2,3-diaminonaphthalene or its derivatives, even under a neutral condition, in the presence of an oxygen source such as dissolved oxygen or oxide compounds (e.g., PTIO and derivatives thereof such as carboxy-PTIO), and a fluorescent naphthalenetriazole or a derivative thereof is obtained. They also found that a method for measuring nitrogen monoxide utilizing the above reaction has extremely high detection sensitivity, and can achieve accurate quantification of very small amount of nitrogen monoxide (see, the specification of Japanese Patent Application No. Hei 7-189978).
However, the aforementioned method utilizing 2,3-diaminonaphthalene requires the irradiation with excitation light having a short wavelength of approximately 370-390 nm for the detection of fluorescence, and this may cause damages to cells and/or tissues in a measurement system. Strong autofluorescence of cells, per se, may also possibly affect the measurement, and moreover, there is a problem that a fluorescence filter provided on a usual fluorescence microscope fails to sufficiently cut off excitation light during fluorescence measurement. In addition, the fluorescent triazole compound formed from 2,3-diaminonaphthalene has rather insufficient fluorescence intensity, and therefore, it is difficult to achieve accurate measurement of intracellular fluorescence of an individual cell by ordinary fluorescence microscopy. Since 2,3-diaminonaphthalene itself has a simple chemical structure, there is also a problem that the compound is not suitable as a fundamental structure for various chemical modifications so as to achieve intracellular localization of an agent.
As a method for measuring nitrogen monoxide that solved these problems, the inventors of the present invention proposed a method utilizing a class of diaminofluorescein derivatives (U.S. Pat. No. 5,874,590). By using the derivatives, nitrogen monoxide can be measured with an excitation light having a long wavelength that gives no damage to living tissues or cells, and intracellularly existing nitrogen monoxide can be accurately measured for each individual cell. The diaminofluorescein derivatives are extremely satisfactory agents from the standpoints of reactivity with nitrogen monoxide and measurement sensitivity. However, the derivatives have a problem that the fluorescence intensity of the triazole derivatives produced through the reaction with nitrogen monoxide slightly increases in the weakly basic to weakly acidic region. Moreover, the triazole compounds also have a problem of instability to light. For these reasons, it has been desired to develop an agent for measurement of nitrogen monoxide that gives no change in fluorescence intensity due to alteration in pH, and produces a fluorescent substance that is stable to light.
An object of the present invention is to provide compounds which are useful for measurement of nitrogen monoxide. More specifically, the object of the present invention is to provide compounds that enable the measurement of nitrogen monoxide by means of excitation light having a long wavelength which does not cause damages to living tissues and cells, and can efficiently react with nitrogen monoxide under a neutral condition to provide a fluorescent substance having excellent fluorescence intensity, which fluorescent substance has stability to light and whose fluorescence intensity is not affected by alteration in pH.
Another object of the present invention is to provide an agent for measuring nitrogen monoxide which comprises a compound having the aforementioned characteristic features. More specifically, the object is to provide an agent for measuring nitrogen monoxide which enables accurate measurement of intracellularly existing nitrogen monoxide for individual cells.
The inventors of the present invention made efforts to achieve the foregoing objects, and as a result, they found that a particular class of fluorescein derivatives, which themselves emit almost no fluorescence, can easily react with nitrogen monoxide under a neutral condition, and give triazole compounds having high fluorescence intensity. They also found that the triazole derivatives can emit strong fluorescence at approximately 515 nm when irradiated with excitation light having a longer wavelength of about 495 nm, and that the derivatives gave substantially no change in fluorescence intensity from weakly basic to weakly acidic regions and the derivatives were stable to light. They further found that intracellular nitrogen monoxide concentrations in individual cells were accurately and conveniently measurable by using these compounds as an agent for measuring nitrogen monoxide. The present invention was achieved on the basis of these findings.
The present invention thus provides a compound represented by the following formula (I): 
wherein R1 and R2 represent amino groups that substitute at adjacent positions on the phenyl ring, provided that either of R1 and R2 represents a mono(C1-6 alkyl)-substituted amino group and the other represents an unsubstituted amino group; and R3 and R4 independently represent hydrogen atom or an acyl group.
According to preferred embodiments of the present invention, there are provided the aforementioned compound wherein the monoalkyl-substituted amino group represented by either of R1 and R2 is a monomethylamino group; and the aforementioned compound wherein both of R3 and R4 represent hydrogen atom.
According to another aspect of the present invention, there is provided an agent for measurement of nitrogen monoxide which comprises the aforementioned compound.
According to further aspect of the present invention, there is provided a compound represented by the following formula (II): 
wherein R11 and R12 combine together to form a group represented by xe2x80x94Nxe2x95x90Nxe2x80x94N(R19)xe2x80x94 which forms a ring structure at adjacent positions on the phenyl ring, wherein R19 represents a C1-6 alkyl group, or R11 and R12 represent a combination of an amino group and a nitro group which substitute at adjacent positions on the phenyl ring; and R13 and R14 independently represent hydrogen atom or an acyl group.
The present invention further provides a method for measuring nitrogen monoxide which comprises the steps of:
(1) reacting a compound represented by the above formula (I) with nitrogen monoxide; and
(2) detecting a compound of the formula (II) formed by the above step (1).